JP5500730B2 - Humanized antibody against β-amyloid peptide - Google Patents

Description

  The present invention relates to the field of antibody technology, more specifically to the field of humanized antibodies to beta-amyloid peptides.

  Alzheimer's disease (AD) is an age-related neurodegenerative disorder, affecting 26.6 million people in 2006. According to forecasts, it is estimated that by 2050, the prevalence will quadruple and by that time 1 in 85 people worldwide will live with the disease (Brookmeyer et al. (2007 )). AD manifests as progressive cognitive deficits, such as memory loss and mental decline.

Central to the pathogenesis of AD is the accumulation of beta-amyloid peptide (Abeta) in the brain. Abeta is a cleavage product of amyloid precursor protein (APP), which is cleaved in turn in the amyloid formation pathway, first by β-secretase and then by γ-secretase. The resulting Abeta fragments are of various sizes, but the 40 amino acid peptide (Abeta ₄₀ ) is the most abundant species, and the 42 amino acid peptide, the so-called Abeta _42, is the most harmful species. It is considered. Abeta can accumulate in the extracellular space of the brain, where it aggregates in a multi-step process to form neurotoxic oligomers, which together with other substances produce amyloid plaques, which are responsible for Alzheimer's disease. It is a typical feature.

  A promising clinical immunological method for the treatment of Alzheimer's disease is passive immunization, in which an antibody against Abeta is administered to a subject to remove Abeta from the brain. Three different mechanisms have been proposed for Abeta clearance by anti-Abeta antibodies, and these are not mutually exclusive: (1) catalytic conversion of fibrillar Abeta to a less toxic form (Bard et al. (2000) Bacskai et al. (2001); Frenkel et al. (2000)); (2) Opsonizing Abeta deposits to induce microglial phagocytosis (Bard et al. (2000); Bacskai et al. (2002) Frenkel et al. (2000); and (3) the so-called peripheral sink hypothesis that promotes Abeta efflux from the brain into the circulation (DeMattos et al. (2001)).

  Mohajera et al. (2004) and Gaugler et al. (2005) at the University of Zurich made mouse antibodies against Abeta and examined the biological activity of monoclonal murine anti-Abeta antibodies in vivo.

  However, murine monoclonal antibodies are often immunogenic when administered to humans. The induced antiglobulin response limits the clinical utility of murine antibodies (Miller et al. (1983); Schroff et al. (1985)).

  Accordingly, there is a need for new non-immunogenic effective antibodies for the treatment and / or diagnosis of Abeta-related disorders, particularly Alzheimer's disease.

Brookmeyer R, Johnson E, Ziegler-Graham K, Arrighi HM (2007): Forecasting the global burden of Alzheimer ’s disease.Alzheimer's & Dementia: The Journal of the Alzheimer's Association (Vol. 3 (3)) Bard, F., Cannon, C., Barbour, R., Burke, RL, Games, D., Grajeda, H., Guido, T., Hu, K., Huang, J., Johnson-Wood, K. , Khan, K., Kholodenko, D., Lee, M., Lieberburg, I., Motter, R., Nguyen, M., Soriano, F., Vasquez, N., Weiss, K., Welch, B. , Seubert, P., Schenk, D. and Yednock, T. (2000): Peripherally administered antibodies against amyloid beta-peptide enter the central nervous system and reduce pathology in a mouse model of Alzheimer disease. Nat. Med. 6, 916 -919 Bacskai, BJ, Kajdasz, ST, Christie, RH, Carter, C., Games, D., Seubert, P., Schenk, D. and Hyman, BT (2001): Imaging of amyloid-beta deposits in brains of living mice permits direct observation of clearance of plaques with immunotherapy. Nat. Med. 7, 369-372 Frenkel, D., Katz, O. and Solomon, B. (2000): Immunization against Alzheimer ’s beta-amyloid plaques via EFRH phage administration. Proc. Natl. Acad. Sci. USA 97, 11455-11459 Bacskai, BJ, Kajdasz, ST, McLellan, ME, Games, D., Seubert, P., Schenk, D. and Hyman, BT (2002): Non-Fcmediated mechanisms are involved in clearance of amyloid-beta in vivo by immunotherapy J. Neurosci. 22, 7873-7878 DeMattos, RB, Bales, KR, Cummins, DJ, Dodart, JC, Paul, SM and Holtzman, DM (2001): Peripheral anti-Abeta antibody alters CNS and plasma Abeta clearance and decreases brain Abeta burden in a mouse model of Alzheimer's disease Proc. Natl. Acad. Sci. USA 98, 8850-8855 Mohajeri, MH, Gaugler, M., Martinez, J., Tracy, J., Li, H., Crameri, A., Kuehnle, K., Wollmer, Am.A. and Nitsch, RM (2004): Assessment of the bioactivity of Antibodies against beta-amyloid peptide in vitro and in vivo. Neurodegenerative dis. 1, pp. 160-167 Gaugler, M., Tracy, J., Kuhnle, K., Crameri, A., Nitsch, RM and Mohajeri, MH (2005): Modulation of Alzheimer's pathology by cerebro-ventricular grafting of hybridoma cells expressing antibodies against Abeta in vivo. FEBS Letters 579, pp. 753-756 Miller, R. et al. (1983): Monoclonal antibody therapeutic trials in seven patients with T-cell lymphoma. Blood 62: 988-995 Schroff, R. et al. (1985): Human anti-murine immunoglobulin responses in patients receiving monoclonal antibody therapy.Cancer Research 45: 879-885

  Accordingly, it is a general object of the present invention to provide antibodies that specifically bind to Abeta, particularly the C-terminal portion of Abeta, and are well tolerated by the human immune system.

In a first aspect, the invention relates to a humanized or fully human form that selectively binds to the C-terminal region of Abeta, particularly between amino acids 30-40 (SEQ. ID. No. 26). An isolated antibody is provided. This antibody exhibits high affinity for both Abeta ₄₂ and Abeta ₄₀ , and further does not substantially recognize amyloid precursor protein (APP) in vivo.

  The advantage of so-called humanized antibodies is that they are generally considered to have minimal or no ability to elicit a response of the human immune system and therefore are considered to be less or less immunogenic when applied to humans. It is to go. Thus, unlike murine antibodies, humanized antibodies are suitable for therapeutic purposes and clinical applications.

  The term “humanization” refers to a well established technique for reducing the immunogenicity of a heterologous antibody. Humanized antibodies are engineered to have as few non-human structures as possible. One method is based on grafting the complementarity determining regions (CDRs) of heterologous antibodies onto the variable light chain VL and variable heavy chain VH of the human acceptor framework. In other methods, the heterologous antibody framework is mutated toward a human framework. In both cases, maintenance of the functionality of the antigen binding portion is essential. To that end, three-dimensional models of the parent sequence and various hypothetical humanized products are analyzed using, for example, computer programs for molecular modeling well known to those skilled in the art. This analysis-especially-can identify framework residues that appear to be directly or indirectly involved in antigen binding. Often, a small number of donor framework residues are important for antigen binding because they are in direct contact with the antigen or because they affect the conformation of a particular CDR (Davies et al (1990 ); Chothia et al (1987)). Thus, if not already present, it is desirable to mutate the corresponding acceptor framework residues towards those donor framework residues identified as important for antigen binding. Humanized antibodies may also comprise residues that are not found in the human germline repertoire in vivo and are not found in the donor CDRs or even in the donor framework.

  The extent of antibody humanization should be indicated by calculating the percent sequence identity of the humanized antibody framework relative to the original human acceptor framework available from the human library used to generate the humanized antibody. Can do. Preferably, the antibodies of the present invention have at least 60% identity to the framework of available human libraries, more preferably at least 75%, at least 80%, at least 85%, at least 90% (in the following order) Most preferably 95% or even 100% of the framework structure. In the concept of the present invention, the term “complementarity determining region” or “CDR” refers to the complementarity determining region consisting of the antigen binding loop of an antibody as defined by Kabat et al. (1991). The CDRs and framework residues of the present invention are determined according to the Kabat definition (Kabat et al. (1987).

  As used herein, the term “antibody” refers to full-length antibodies, such as monoclonal antibodies, and any antigen-binding fragment or single chain thereof that has sufficient binding capacity for the selected antigen. Examples of antigen binding fragments included in the present invention include Fab fragments, F (ab ′) 2 fragments, Fd fragments, Fv fragments; single domain or dAb fragments, isolated complementarity determining regions (CDRs); A combination of two or more isolated CDRs, optionally linked by a synthetic linker, and a single chain variable fragment (scFv) are included. “Full-length antibodies” include chimeric antibodies in which one antigen-binding variable domain is bound to a different derived constant domain; for example, the murine antibody variable domain Fv is a human antibody constant domain. It is bound to Fc.

The antibody fragments listed above are obtained using conventional methods known to those skilled in the art, and those fragments are screened for utility in the same manner as intact antibodies.
In the present invention, the CDR is derived from the monoclonal mouse antibody 22C4 (Mohajeri et al. (2002), J. Biol. Chem. 277, pp. 33012-33017, and Neurogenegenerative Dis. 1 (2004), pp. 160-167. ). This murine antibody is directed against the C-terminal portion of Abeta, more specifically an epitope within amino acids 30-40 (SEQ. ID. No. 26).

In a preferred embodiment, the antibodies block their oligomerization by binding to the target of Abeta, in particular Abeta ₄₀ and / or Abeta ₄₂ .
The present invention relates to SEQ ID: No. 1, SEQ ID: No. 2, SEQ ID: No. 3, SEQ ID: No. 4, SEQ ID: No. 5 and SEQ ID: No. An antibody comprising one or more complementarity determining region (CDR) sequences having at least 80% identity to a group of 6 sequences is provided.

  As already mentioned, the CDR of the present invention, ie, SEQ ID: No. 1, SEQ ID: No. 2, SEQ ID: No. 3, SEQ ID: No. 4, SEQ ID: No. 5 and SEQ ID: No. 6 can be grafted into a suitable acceptor framework. The term “framework” refers to the art-recognized portion of an antibody variable region that lies between the more diverse CDR regions. Such framework regions are commonly referred to as framework structures 1-4 (FR1, FR2, FR3, and FR4) and retain the three CDRs in the heavy and light chain variable regions of the antibody in three-dimensional space. Provides a scaffold for the CDRs to form an antigen binding surface. Suitable acceptor frameworks are preferably immunoglobulin-derived antigen binding polypeptide frameworks well known in the art, including but not limited to: VhH domain, V-NAR domain, Vh domain, Fab, scFv, bis-scFv, camel IG, IfNAR, IgG, Fab2, Fab3, minibody, diabodies, triabodies, and tetrabodies (Ref: Holliger, P. and Hudson, P. (2005), Nat. Biotechnol. 23 (9), pp. 1126-1136). The framework sequence may be a human consensus sequence.

  The antibody is selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and isoforms, and can include more than one class or isoform sequence.

  Preferably, the antibody is at least 60% identical to the framework of an available human library, more preferably at least 75%, at least 80%, at least 85%, at least 90%, most preferably (in the following order) Includes framework structures with 95%, or even 100% identity.

  Since CDRs can be grafted onto human frameworks and the resulting antibodies contain non-human CDRs and human or essentially human frameworks, the antibodies of the invention are recombinant molecules. Alternatively, the antibody is derived from a non-human antibody and its framework is mutated towards the human antibody. Both variants are included in the term “available human library”.

In one embodiment of the invention, the antibody is an scFv antibody. scFv is a short linker peptide, such as a linker containing 1-4 repeats of the sequence GGGGS, preferably (GGGGGS) ₄ peptide (SEQ ID No. 25), or Alfthan et al. (1995) Protein Eng. 8: 725- It may be a full-length scFv comprising VL and VH domains linked by the linker disclosed in 731, or simply a VL or VH domain that has sufficient binding capacity for the selected antigen. The linkage of VL and VH may be either VL-linker-VH or VH-linker-VL orientation.

  In one embodiment, the scFv framework is stable and soluble in a reducing environment. These characteristics can be confirmed by a so-called quality control system disclosed in WO01 / 48017. The stability of these antibodies is preferably at least about half as good as that of particularly stable lambda grafts, more preferably at least as good as lambda grafts, and most preferably better than lambda grafts. Woern et al. (2000) describes that the onset of lambda graft denaturation is approximately 2.0 M GdnHCl. It has been shown that scFvs that perform well in quality control systems are stable and soluble even under oxidizing conditions. In a preferred embodiment, the solubility of the antibody of the invention as measured according to the method of Atha and Ingham (1981) is at least 5 mg / ml, more preferably at least 10 mg / ml, most preferably at least 20 mg / ml.

In other preferred embodiments, the antibody comprises SEQ ID. No. 7, SEQ ID. No. 8, SEQ ID. No. 10, SEQ ID. No. 11, SEQ ID. No. 12, SEQ ID. No. 13, SEQ ID. No. 14, SEQ ID. No. 15 and SEQ ID. No. It comprises a variable light chain fragment (V _L ) framework that is identical to or derived from the framework sequence contained in the group of 16 sequences. In the case of a derived sequence, the sequence is SEQ ID. No. 7, SEQ ID. No. 8, SEQ ID. No. 10, SEQ ID. No. 11, SEQ ID. No. 12, SEQ ID. No. 13, SEQ ID. No. 14, SEQ ID. No. 15 and SEQ ID. No. At least 60% identity to a group of 16 sequences, more preferably (in the following order) at least 75%, at least 80%, at least 85%, at least 90%, most preferably 95%, or even 100% Shows the identity.

In other preferred embodiments, the antibody comprises SEQ ID. No. 17, SEQ ID. No. 18, SEQ ID. No. 20, SEQ ID. No. 21, SEQ ID. No. 22 and SEQ ID. No. It includes a variable heavy chain fragment (V _H ) framework that is identical to or derived from the framework sequence contained in the group of 23 sequences. In the case of a derived sequence, the sequence is SEQ ID. No. 17, SEQ ID. No. 18, SEQ ID. No. 20, SEQ ID. No. 21, SEQ ID. No. 22 and SEQ ID. No. At least 60% identity to a sequence of groups consisting of 23, more preferably (in the following order) at least 75%, at least 80%, at least 85%, at least 90%, most preferably 95%, or even 100% Shows the identity.

  Sequence SEQ ID. No. 8, SEQ ID. No. 10, SEQ ID. No. 11, SEQ ID. No. 12, SEQ ID. No. 13, SEQ ID. No. 14, SEQ ID. No. 15, SEQ ID. No. 16, SEQ ID. No. 20, SEQ ID. No. 21, SEQ ID. No. 22 and SEQ ID. No. 23 is disclosed in WO 03/097697. These framework sequences are derived from human immunoglobulin origin and their solubility and stability properties under reducing conditions have been demonstrated in the so-called quality control system disclosed in WO 01/48017.

More preferably, the antibody is SEQ ID. No. 7 VH fragment and SEQ ID. No. Contains 17 VL sequences.
Most preferably, the antibody is Seq. ID. No. It has a sequence that is at least 60% identical to 24, more preferably at least 75%, 80%, 90%, 95% identical. In the most preferred embodiment, the structure of the antibody of the present invention is Seq. ID. No. 24. In this antibody, SEQ ID. No. 7 and SEQ ID. No. 17 are linked by a (GGGGGS) ₄ linker. The obtained scFv antibody was named ESBA212.

  Those skilled in the art will appreciate that the sequences of the present invention can be altered such that the amino acid sequence differs from the sequences disclosed herein while maintaining the selective binding ability to the C-terminal portion of Abeta. Will. Thus, neither the framework of the humanized antibody nor the CDR regions need to closely match the donor CDR or acceptor framework. Changes in them can be made by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of the antibody by standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Such mutations can be introduced for various purposes, for example to improve the binding, solubility or stability properties of the antibody. The antibodies of the present invention can also contain conservative amino acid substitutions in one or more non-essential amino acid residues. In other embodiments, mutations can be introduced randomly, eg, by saturation mutagenesis, into the entire or part of the coding sequence and the resulting mutants screened for their ability to bind the target of interest.

  The percent identity between two sequences is a function of the number of identical positions shared by those sequences, taking into account the number of gaps that need to be introduced to obtain an optimal alignment of the two sequences and the length of each gap. . Comparison of sequences between two sequences and determination of percent identity can be accomplished using mathematical algorithms, which are well known to those skilled in the art. The identity described herein is the BLAST program accessible via the Internet (Basic Local Alignment Search Tools; see: Altschul, SF, Gish, W., Miller, W., Myers, EW & Lipman, DJ (1990). "Basic local alignment search tool." J. Mol. Biol. 215: 403-410). A BLAST protein search can be performed by comparing the amino acid sequence with the protein molecule of the present invention using the XBLAST program, score = 50, language length = 3. To obtain a gapped alignment for comparison, Gapped BLAST as described in Altschul et al., (1997) Nucleic Acids Res. 25 (17): 3389-3402 can be used. When utilizing BLAST and Gapped BLAST programs, the default parameters of each program (eg, XBLAST and NBLAST) can be used.

  The protein sequence of the present invention can be further used as a “query sequence” to perform a search against a public database to identify, for example, related sequences. Such a search can be performed using the XBLAST program (version 2.0) described above.

Humanized or fully human antibodies included in the present invention exhibit the following properties:
(I) binds to the C-terminus of Abeta and thus binds to both Abeta ₄₀ and Abeta ₄₂ with high and substantially equal affinity;
(Ii) high affinity for oligomeric and monomeric forms of Abeta;
(Iii) substantially does not recognize amyloid precursor protein (APP) in vivo;
(Iv) has a solubility of at least 5 mg / ml, preferably at least 10 mg / ml, more preferably at least 20 mg / ml; and (v) exhibits low or no immunogenicity when applied to humans Not shown.

Furthermore, the antibodies of the present invention preferably exhibit at least one of the following properties, more preferably more than one and most preferably all:
(Vi) mediates the uptake of fibrillar Abeta by microglia;
(Vi) bind beta-amyloid plaques;
(Vii) removing beta-amyloid plaques in the brain and / or preventing the formation of amyloid plaques in the brain;
(Viii) reduce neuronal vulnerability associated with Abeta toxicity and pro-toxic events caused by seizures;
(Ix) cross the blood brain barrier; and / or (x) substantially restore normal behavior;
(Xi) Remove beta-amyloid fibrils in the brain and / or prevent the formation of amyloid fibrils in the brain.

  The antibodies of the invention do not substantially recognize amyloid precursor protein (APP) in vivo. Preferably, the binding affinity for Abeta is at least 2-fold, more preferably at least 5-fold, even more preferably at least 10-fold, particularly preferably at least 50-fold, most preferably at least when compared to the binding affinity for APP. 100 times higher.

  Thus, when the antibody of the invention is administered to a subject, APP does not compete with Abeta for binding, and this antibody does not interfere with the biological properties of uncleaved APP. This feature is of particular interest, for example, for diagnostic purposes and / or medical treatment of neurological disorders associated with abnormal Abeta accumulation and / or deposition in the central nervous system.

  As used herein, the term “neuropathy” includes, but is not limited to, Alzheimer's disease, mild cognitive impairment, aphasia, frontotemporal dementia, Lewy body disease, Parkinson's disease, Pick's disease, Binswangel disease, cerebral amyloid angiopathy, Down syndrome, multiple infarct dementia, Huntington's disease, Creutzfeldt-Jakob disease, AIDS dementia complications, depression, anxiety disorder, phobia, Bell's palsy, epilepsy Encephalitis, multiple sclerosis; neuromuscular disorders, neurotumor disorders, brain tumors, neurovascular disorders; including stroke, neuroimmune disorders, neurootology, neurotrauma: including spinal cord injury, pain: including neuropathic pain Pediatric neuropathy and neuropsychiatric disorder, sleep disorder, Tourette syndrome, mild cognitive impairment, vascular dementia, multiple infarct dementia, cystic fibrosis, Gaucher disease Other motor disorders, glaucoma, and central nervous system (CNS) diseases in general. More preferably, the antibodies of the invention are used for the treatment, prevention, progression delay or diagnosis of Alzheimer's disease, stroke, neurotrauma and glaucoma.

  In other aspects, the antibodies of the invention are chemically modified. Chemical modification can alter antibody properties such as stability, solubility, antigen binding specificity or affinity, in vivo half-life, cytotoxicity, and tissue permeability. Chemical modifications are well known to those skilled in the art. A preferred chemical modification of the antibodies of the invention is PEGylation.

In one embodiment, the antibody is conjugated to a therapeutic agent, such as a toxin or chemotherapeutic compound. Antibody to a radioisotope, such as but not limited to ²¹² Bi, ¹²⁵ I, ¹³¹ I, ⁹⁰ Y, ⁶⁷ Cu, ²¹² Bi, ²¹² At, ²¹¹ Pb, ⁴⁷ Sc, ¹⁰⁹ Pd and ¹⁸⁸ Re, eg, immunotherapy Can be conjugated.

In yet other embodiments, the antibodies of the invention can be linked to a label. The label can enable colorimetric detection of the antibody. Alternatively, the antibody is radiolabeled. Most preferably, the radioactive label is ⁶⁴ Cu.

Another object of the present invention is to provide a diagnostic or scientific tool comprising the antibodies disclosed herein.
The antibodies of the invention can be used in diagnosing or screening a subject for amyloidosis or Alzheimer's disease, or in determining a subject's risk for the development of amyloidosis or Alzheimer's disease.

  In yet another embodiment, the present invention further includes a diagnostic method comprising administering an effective amount of an antibody of the present invention to a subject, preferably a mammal. The method further includes detecting the label.

  In yet other embodiments, the invention includes an immunoassay comprising an antibody described herein, wherein the assay may be either an in vivo or in vitro immunoassay. The antibody can be used in liquid phase or bound to a solid phase. Examples of such immunoassays include radioimmunoassay (RIA), flow cytometry, western blot and microarray.

Furthermore, the present invention includes a test kit comprising the antibody disclosed herein.
In a preferred embodiment, the antibodies of the present invention mediate fibrillar Abeta uptake by microglia, thereby reducing Abeta levels in vivo.

In yet another preferred embodiment, the antibody of the invention improves cognitive behavior and reduces the number of immature neurons when administered in an effective amount in a subject with Alzheimer's disease.
The present invention further discloses herein for treating, preventing and / or delaying progression of neuropathy or amyloidosis characterized by abnormal accumulation and / or deposition of Abeta in the central nervous system, in particular Alzheimer's disease. A pharmaceutical composition containing the antibody is included.

  In yet another embodiment, the present invention produces a pharmaceutical composition for the treatment, prevention and / or delay of progression of said neurological disorders, preferably Alzheimer's disease and / or for passive immunization against Alzheimer's disease. A method is provided comprising the step of combining an antibody disclosed herein with at least one suitable pharmaceutical carrier.

Preferably, the pharmaceutical compositions disclosed herein inhibit and / or reduce the effects of Abeta accumulation in the subject's brain.
The antibodies of the invention can be administered in combination with a pharmaceutically acceptable carrier or in combination with one or more other active agents. The active agent may be a small organic molecule and / or an anti-Abeta antibody.

  The antibodies and / or pharmaceutical compositions disclosed herein can be obtained in various ways, for example, intravenous, intraperitoneal, intranasal, subcutaneous, intramuscular, topical or intradermal, intracranial, subarachnoid in cerebrospinal fluid. Can be administered. Preferred types of application are (in this order) intranasal, subcutaneous, intravenous, intrathecal cerebrospinal fluid, and intracranial.

  Commonly used formulations are known to those skilled in the art. For example, aerosol formulations, such as nasal spray formulations, include purified aqueous solutions and other solutions of active agents, including preservatives and isotonic agents. Such formulations are preferably adjusted to a pH and isotonic state compatible with the nasal mucosa.

  Furthermore, simultaneous or sequential administration of other drugs is desirable. Preferably, the antibodies of the invention are present in an amount sufficient to restore normal behavior and / or cognitive characteristics in a case of Alzheimer's disease.

  In yet another embodiment, the present invention provides a method for treating, preventing and / or delaying progression of the neurological diseases described above, comprising administering an effective amount of an antibody of the present invention to a subject in need thereof. A method comprising:

  Another object of the present invention is to provide a method for passive immunization of a mammal comprising the step of administering to the mammal an antibody disclosed herein. Preferably, passive immunization is performed within anti-Abeta immunotherapy.

  In another aspect, the invention features an isolated nucleic acid sequence that includes a sequence that encodes an amino acid sequence included in the invention. Such a nucleic acid sequence may be either DNA or RNA, and may be either single-stranded or double-stranded.

The present invention further provides a cloning or expression vector comprising a DNA sequence encoding a polypeptide of the present invention, most preferably an antibody.
In addition, suitable host cells harboring vectors and / or nucleic acid sequences comprising sequences encoding the amino acid sequences disclosed herein are provided. This may be a prokaryotic or eukaryotic cell, in particular an E. coli, yeast, plant, insect or mammalian cell.

  The antibodies of the present invention can be made using routine techniques in the field of recombinant molecular biology. Knowing the sequences of the polypeptides, one skilled in the art can generate corresponding cDNAs encoding those polypeptides by gene synthesis.

  In another embodiment, a method for producing the antibody of the present invention, comprising culturing a host cell transformed with DNA encoding the antibody under conditions capable of synthesizing the antibody, and culturing the molecule in a culture. There is provided a method comprising recovering from the process. Preferably, the method provides scFv antibodies purified from E. coli inclusion bodies or from the E. coli periplasm if the scFv construct used contains a signal sequence that directs the polypeptide to the periplasm. It may be necessary to include a reconstitution step to refold the antibody into a functional molecule.

  In yet another aspect, the invention provides a method of treatment comprising administering to a subject in need thereof a therapeutically effective amount of a polynucleotide, vector or host cell described herein.

In a second aspect, the present invention provides a diagnostic method comprising the following steps:
(I) labeling the antibody;
(Ii) administering an effective amount of the antibody to the subject intranasally or systemically; and (iii) detecting the concentration and / or presence of labeled antibody in a body part of the subject.

The antibody is a humanized antibody of the present invention, preferably a single chain antibody (scFv), against an epitope formed within amino acids 30-40 of Abeta. In a preferred embodiment, the antibody is labeled with a positron emitting isotope, most preferably ⁶⁴ Cu.

  The term “effective amount” as used herein refers to an amount sufficient to reach or at least partially reach a desired effect, eg, a detectable signal. The effective amount for this application will depend on the detection intensity of the label, the weight of the subject, and the size of the area to be examined.

Preferably, the subject is a mammal; more preferably, the subject is a human.
Positron emission tomography (PET), a medical imaging technique that creates a three-dimensional image of a body part, is based on the detection of radiation by positron emission. In general, a biomolecule is radiolabeled; for example, it incorporates a radiotracer isotope. When this labeled biomolecule is generally administered to a subject by injection into the blood circulation, the radiolabeled biomolecule is concentrated in the target tissue. The object is then placed in an imaging scanner, which detects positron emission.

In one embodiment, step iii) is performed by administering a ⁶⁴ Cu labeled antibody to the subject and placing the subject in an imaging scanner to detect positron emission.
Accordingly, the present invention includes a method for PET imaging comprising administering to a subject a ⁶⁴ Cu labeled antibody of the present invention.

  The sequences of the present invention are:

  Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not limiting.

  It is understood that various aspects, preferred forms and ranges can be arbitrarily combined. Further, depending on the specific aspect, the selected definition, aspect or range may not apply.

In view of the following detailed description of the invention, the invention will be better understood and objects other than those set forth above will become apparent. Such description refers to the accompanying drawings.
FIG. 1 shows a sequence comparison between the light chain variable region of ESBA212 and the VL of ESBATtech framework 2.3 grafted with 22C4-derived Abeta-specific CDRs. FIG. 2 shows a sequence comparison between the heavy chain variable region VH of ESBA212 and the VH of ESBATtech framework structure 2.3. FIG. 3 shows a sequence alignment of the VH of ESBA212 and the original VH derived from the mouse 22C4 antibody. FIG. 4 shows a sequence alignment of ESBA212, 22C4 and framework VH. FIG. 5 shows the results of ex vivo labeling of plaques in an AD mouse model with ESBA212. Either paraffin sections or frozen sections (unfixed and post-fixed) were used. 5A: Mouse model SwePS1, paraffin section; FIG. 5B: Mouse model SwePS1, frozen section; FIG. 5C: Mouse model SweArc, paraffin section; FIG. 5D: Mouse model SweArc, frozen section. FIG. 6 shows the results of ex vivo labeling of plaques in human AD brain with ESBA212. Either paraffin sections or frozen sections (unfixed and post-fixed) were used. FIG. 6A: Paraffin sections; FIG. 6B: Freeze, acetone fixed; FIG. 6C: Freeze, untreated; FIG. 6D: Freeze; Acetone fixed, boiled in citrate buffer for 10 minutes. FIG. 7 shows the results of ex vivo staining of amyloid plaques in brain sections of SwePS1 mice with ESBA212 (FIG. 7A) and thioflavin S (FIG. 7B). FIG. 8 shows the size exclusion chromatography results of ESBA212 bound to FITC-labeled Abeta42 monomer (FIG. 8A), and FIG. 8B shows a control in which framework FW2.3 was incubated with FITC-labeled Abeta42 monomer. . FIG. 9 shows brain homogenates of transgenic SwePS1 mice as antigens (FIG. 9A; column 1: ESBA212, column 2: 6E10) and ADDLs (FIG. 9B; column 1: Fw2.3, column 2: ESBA212, column 3: 6E10). 2 shows two Abeta42 immunoblots using. ESBA212 mainly recognizes monomers and weakly recognizes trimers. FIG. 10 shows the measurement of the affinity of ESBA212 by ELISA (FIG. 10A: for Abeta40; affinity constant: 6.26 nM; and FIG. 10B: for Abeta42; affinity constant: 6.31 nM). FIG. 11 shows mass spectrometry of ESBA212. FIG. 12 represents the FT-IR spectrum and shows the percent unfolding of ESBA212 at various temperatures ranging from 25-95 ° C. FIG. 13 is a Western blot showing the stability of ESBA212 incubated in the presence (row 1) or absence (row 2) of mouse brain homogenate. FIG. 14 is the result of a thioflavin T assay and shows that ESBA212 inhibits Abeta42 oligomerization in vitro. Figure 15 shows ESBA212 mean serum concentration over time after a single injection intravenously or intraperitoneally. FIG. 16 shows detection of bound ESBA212 after intranasal (FIG. 16A) or intravenous (FIG. 16B) administration. ESBA212 binds to amyloid plaques in the brain regardless of the chosen route of administration. FIG. 17 shows detection of bound ESBA212 in the brains of SwePS1 mice after intranasal administration (FIG. 17A) and after thioflavin S staining (FIG. 17A), confirming that ESBA212 binds to amyloid plaques. FIG. 18 is an ex vivo labeling of amyloid plaques with ⁶⁴ Cu-ESBA212 (FIGS. 18B and D) compared to ESBA212 (FIGS. 18A and C). 18A and B show paraffin sections, while FIGS. 18C and D show frozen sections. FIG. 19 shows the detection of ESBA212 and Cu-ESBA212 bound to mouse (SwePS1) brain sections 24 and 48 hours after intranasal administration. Figure 19A: ESBA212 after 24 hours; Figure 19B: ESBA212 after 48 hours; Figure 19C: Cu-ESBA212 after 24 hours; and Figure 19D: Cu-ESBA212 after 48 hours. FIG. 20 shows the detection of ESBA212 in the kidney after intravenous ESBA212 injection. FIG. 21 shows yet another intranasal administration similar to FIG. 16A, showing Aβ staining in fixed SwePS1 mouse brain sections of animals perfused for 1 hour after intranasal treatment with 200 μg ESBA212; FIG. 21A: anti-His antibody FIG. 21B: control stained with 6E10; FIG. 21C: negative control stained with anti-rabbit Cy3; FIG. 21D: control stained with 22c4 IgG. FIG. 22 shows brain Aβ40 and 42 levels in APPswe / PS1ΔE9 mice treated with PBS, 22c4 IgG and ESBA212 for 3 months. FIG. 22A: soluble fraction (TBS); FIG. 22B: insoluble fraction (GuHCl); FIG. 22C: membrane bound fraction (TBS-Triton). FIG. 23 shows paraffin sections of treated APPswe / PS1ΔE9 mice stained with 6E10 and evaluated by ImageJ software; FIG. 23A: number of plaques; FIG. 23B: plaque area.

Example 1: Production of ESBA212 The antigen binding site of single chain antibody ESBA212 was obtained from murine antibody 22C4 and was identified by the University of Zurich. Abeta-specific mouse IgG antibody 22C4 is formed by immunizing mice with Abeta _30-42 and is therefore directed to the C-terminus of Abeta. Cloning of the VL and VH domains was performed by RT-PCR according to Burmester and Plueckthun (2001). In summary, the mRNA was obtained from hybridoma cells producing antibody 22C4. RT-PCR using primers described by Burmester and Plueckthun was performed to amplify the VL and VH domains. These two domains were assembled by SOE-PCR (splicing by overlap extension; splicing by overlap extension). The amplified single chain variable region fragment (scFv) was then digested with SfiI, cloned into an appropriate expression vector and sequenced. Thus, a mouse scFv fragment was obtained that maintained its specificity for Abeta ₄₂ . This scFv was humanized to obtain a single chain antibody ESBA212 (see FIGS. 1-4 for sequence comparison. FIGS. 1 and 2 show the framework structure in which ESBA212 and 22C4-derived Abeta-specific CDRs were grafted. Alignment with 3).

  A plasmid encoding ESBA212 was introduced into an appropriate E. coli strain (eg, BL21) and expressed as inclusion bodies. Inclusion body refolding followed by purification by gel filtration yielded functional single chain antibodies.

Example 2: Binding to amyloid plaques on tissue sections To test whether scFv ESBA212 can recognize Abeta, ex vivo immunostaining of brain tissue containing amyloid plaques was performed. For this, brain tissue sections from various transgenic mice (SweArc, SwePS1) that express human APP and form Abeta plaques, and brain sections from human Alzheimer's disease patients were used.

  ESBA212 reacted with amyloid plaques in both frozen and paraffin sections from mice, regardless of the Alzheimer's disease mouse model used (FIG. 5). ESBA212 also stained plaques on fixed human Alzheimer's disease tissue (FIG. 6A). Especially around blood vessels, strong staining known as amyloid angiopathy was observed. Amyloid angiopathy refers to the deposition of beta-amyloid in cerebral cortex and buffy coat small and medium sized vessels. However, ESBA212 does not bind to amyloid plaques on human frozen sections in either unfixed (FIG. 6B) or post-fixed (FIGS. 6C, D) sections.

  These different results between human and rodent sections can be explained by the possibility that the amount of various plaques differs compared to humans because transgenic mice overexpress human APP. Guentert et al. (2006) describe the ultrastructural differences between the transgenic mouse model and the scattered plaques in the brain of human AD. In the mouse brain, dense plaques of various sizes dominate. In the cortex and hippocampus of human AD patients, this type of plaque occurs only in 10%, whereas cored plaque dominates (about 90%) (Guentert et al., 2006). Due to these differences observed, the C-terminus can be reached in the brain of transgenic mice, but not in the brain of AD patients. Fixation of human brain tissue may have altered the conformation of amyloid plaques so that ESBA212 can be reached by exposing the embedded C-terminal epitope otherwise.

  FIG. 7 shows the specific binding of ESBA212 to amyloid plaques on SwePS1 brain sections as an ESBA212 staining pattern consistent with the pattern of thioflavin S, a standard fluorescent dye that specifically binds to amyloid protein deposits. .

Example 3 Binding to Abeta42 Monomer It was possible to demonstrate that ESBA212 binds to FITC-labeled Abeta42 by size exclusion chromatography. ESBA212 was incubated with FITC-Abeta42 and loaded onto the column. ESBA212 and FITC-Abeta42 elute together exactly as a peak for ESBA212 and a peak for FITC-Abeta42 (FIG. 8A) and also show a 5 kDa size shift compared to ESBA212 alone (data not shown) Therefore, it represents the bound FITC-Abeta42 (5 kDa). However, when FITC-Abeta42 is incubated with framework FW2.3 that contains the same framework region as ESBA212 but does not contain Abeta-specific CDRs, it clearly distinguishes two peaks: a peak for scFv and a second peak for FITC-Abeta42. Peak was seen (FIG. 8B).

Example 4: Binding to different Abeta42 conformations To further elucidate the specificity of ESBA212, Abeta42 immunoblots were performed. To this end, brain homogenates of SwePS1 mice and in vitro prepared ADDLs (amyloid beta-diffused ligands; amyloid beta-derived diffusible ligands; protocol by Klein) were separated on SDS gels and transferred to membranes. Abeta was detected using ESBA212 and 6E10 (Abeta specific IgG as a control). Control antibody 6E10 also recognized Abeta42 monomer, beta-stubs, trimer, and full-length APP. However, ESBA212 mainly recognized Abeta42 (FIG. 9).

Example 5: Elucidation of affinity in vitro The binding properties of ESBA212 were examined in an ELISA using Abeta40 and Abeta42 as antigens, respectively. 96-well plates were coated with 5 ug / ml neutravidin in dilution buffer (PBS / 0.01% BSA / 0.2% Tween-20) and incubated overnight at 4 ° C. The plates were then washed 3 times with TBS / 0.005% Tween-20. Biotinylated Abeta 40 or Abeta 42 (1 ug / ml in dilution buffer) was added to each well and the plates were incubated at room temperature for 15 minutes. Plates were washed as above and non-specific binding sites were blocked by the addition of dilution buffer. Plates were incubated for an additional 1.5 hours at room temperature with shaking. After washing, ESBA212 diluted solution prepared at 0 to 500 nM was added to the wells by the triple test method and incubated at room temperature (RT) with shaking for 1 hour. Plates were washed and ESBA212 was detected with purified rabbit polyclonal anti-ESBA212 antibody (1: 10000 in dilution buffer) for 1 hour at room temperature. After washing, horseradish peroxidase-labeled anti-rabbit IgG (1: 4000 in dilution buffer) was used to detect the previously bound rabbit antibody, and POD was used as a substrate. The reaction was stopped by the addition of 1M HCl and the absorbance at 450 nm was read.

  The EC50 could be calculated from the measured ESBA212 curve. For ESBA212, the EC50 was determined to be in the range of 1-15 nM for both Abeta40 and Abeta42 (FIG. 10).

Example 6: Mass measurement The exact mass of ESBA212 was measured by electrospray mass spectrometry. To that end, ESBA212 was purified and measured in 50% acetonitrile / 0.2% formic acid (pH 2). The mass spectrum (neutral mass) was deconvoluted using MaxEnt1 software.

The mass of ESBA212 was determined to be 26517 Da (FIG. 11).
Example 7: Solubility Measurement by PEG Precipitation The apparent solubility of ESBA212 was measured in the presence of polyethylene glycol 3000 (PEG 3000) according to the method of Atha and Ingham (1981). ESBA212 (20 mg / ml) was incubated with equal volumes of buffer containing various concentrations of PEG3000 (30-50%) to a final protein concentration of 10 mg / ml and a final PEG concentration of 15-25%. After 30 minutes incubation at room temperature, the sample was centrifuged and the protein concentration in the supernatant was determined from the absorbance at 280 nm. Apparent solubility was calculated by plotting the PEG concentration against the logarithm of the protein concentration measured in the supernatant. Solubility was determined by extrapolating to 0% PEG.

The solubility of ESBA212 was determined to be about 20 mg / ml.
Example 8: Measurement of thermal stability The thermal stability of ESBA212 is determined by measuring a Fourier Transform-Infrared (FT-IR) spectrum as the temperature is increased from 25 ° C to 95 ° C with a Bruker Sensor 27 spectrometer. It was judged by. Prior to measuring the spectrum, ESBA212 was allowed to equilibrate at each temperature for 1-2 minutes.

  The melting temperature of ESBA212 (protein unfolding 50%) was determined to be 62.8 ° C. However, ESBA212 unfolding already starts at about 40 ° C. (FIG. 12).

Example 9: Inhibition of Abeta Aggregation In Vitro It was investigated whether ESBA212 can inhibit the formation of Abeta42 oligomerization. To that end, the thioflavin T assay was performed in solution. Thioflavin T rapidly associates with aggregated Abeta fibrils and enhances emission at 482 nm, unlike the free dye 445 nm. This change is dependent on the aggregated state of Abeta since monomeric or dimeric peptides do not react (LeVine III, 1993).

  2.5 μM Abeta42 was incubated in the presence of Thioflavin T and 500 mM NaCl with or without 2.27 μM ESBA212. As clearly seen in FIG. 14, the presence of ESBA212 clearly blocks Abeta oligomerization for more than 3 hours in vitro, as the fluorescence measured at 482 nm does not increase. In contrast, higher levels of fluorescence are measured when ESBA212 is not added to the solution, which is an indicator of the formation of Abeta aggregates.

Example 10 In Vivo Elucidation of Pharmacokinetics To determine the pharmacokinetics of ESBA212, APP-transgenic mice and non-transgenic littermates were treated with a single intravenous or intraperitoneal injection of ESBA212. Blood was collected retro-orbitally at a predetermined time after injection, the amount of ESBA212 in the serum was measured by quantitative ELISA, and the data was analyzed by PK-Sumit software.

  Animals: Alzheimer's disease mouse models produced by Prof. Dr. R. Nitsch (University of Zurich) were used for pharmacokinetic experiments. These mice (PrP-APP Sw / Arc 10+) overexpress human APP695 containing both Swedish and Arctic mutations under the control of the prion protein promoter in a single construct (Knobloch et al, 2006). These mice were bred on a C57BL / 6 and DBA / 2 hybrid background to prevent development of health and reproductive problems. The characteristics of this line are: i) Early intracellular Abeta deposits associated with cognitive impairment are formed from 6 months of age; ii) Plaque formation begins at 7 months of age and then over several months Increase significantly. The development of this beta-amyloid plaque is consistent with severe cerebral amyloid angiopathy (CAA) (Knobloch et al. 2006).

The mice used for the pharmacokinetic study were 6 months old.
Experimental method: Pharmacokinetic parameters were determined after intravenous or intraperitoneal injection of ESBA212. For intravenous treatment, APP-transgenic mice and non-transgenic littermates were used. Two groups of 7 animals received a single intravenous injection of 15 mg / kg ESBA212 in PBS (pH 6.5). At predetermined time points (10 and 30 minutes, 1, 2, 4, 8, 12 and 24 hours), blood was collected retro-orbitally by taking 4 samples at each time point (exception: 10 minutes and Only 2 samples at 24 hours). Serum ESBA212 concentration was measured by quantitative ELISA.

  For intraperitoneal application of ESBA212, only APP-transgenic mice were used. Two groups of 7 animals received a single intraperitoneal injection of 20 mg / kg ESBA212 in PBS (pH 6.5). Blood samples were collected as described above for intravenous treatment.

  Results: Systemic pharmacokinetics followed two-phase clearance after intravenous injection of ESBA212. Upon injection, a clear distribution phase (α-excretion, 0 to 1 hour) and terminal elimination phase (β-excretion, 8 to 12 hours) were seen (FIG. 15). Significant in alpha-excretion half-life (0.26 hours vs. -0.23 hours) and terminal half-life (8.79 hours vs. -7.11 hours) between transgenic and non-transgenic mice, respectively There was no difference (Table 1). Other calculated pharmacokinetic values were comparable in transgenic and non-transgenic mice. However, the observed volume of distribution (Vd) is quite high (77.69 and 76.72 ml). This appears to suggest that ESBA212 penetrates into the tissue very well and quickly. In contrast, when ESBA212 was applied intraperitoneally, a difference in pharmacokinetics was observed. A clear absorption phase with peak ESBA212 serum concentration could be detected 1 hour after injection. The distribution half-life was then about 5 times longer than after intravenous application; this would be an indication that a reservoir effect would occur when injected by the intraperitoneal route. However, the calculated terminal half-life was slightly shorter after intraperitoneal injection than intravenous injection. A clear difference was also detected in the mean residence time (MRT) of ESBA212 in vivo, which was about 3 times longer when scFv was injected intraperitoneally rather than intravenously. The observed differences in AUC (area under the curve), distribution volume (Vd) and clearance (CL) between the intravenous and intraperitoneal routes were that higher doses of ESBA212 were administered to intraperitoneally treated animals. This can be explained by the fact that (20 mg / kg instead of 15 mg / kg). Calculation of the pharmacokinetic parameters for an intraperitoneal dose of 15 mg / kg based on the values obtained for 20 mg / kg yielded the following parameters: changes in absorption, distribution and half-life of elimination and MRT Absent. However, at that time, AUC (79.124 ug-h / ml), Vd (67.52 ml) and CL (7.84 ml / h) were equivalent to the values obtained after intravenous injection.

Example 11: Marking of plaques in vivo It was investigated whether ESBA212 could bind to amyloid plaques when applied intravenously or intranasally.

  To that end, transgenic SwePS1 mice were treated with 210 μg ESBA212 three times every 24 hours, either by intravenous or intranasal application route. The animals were perfused with PBS 72 hours after the first application and the brain was analyzed for the presence of ESBA212. ESBA212 bound to brain amyloid plaques after both intravenous or intranasal application of scFv (FIG. 16). Thioflavin S staining confirmed that ESBA212 actually bound to amyloid plaques. Serial brain sections of SwePS1 mice treated intranasally with ESBA212 showed the same staining pattern for ESBA212 and thioflavin S (FIG. 17).

Example ^12: 64 ⁶⁴ Cu-labeled PET imaging ESBA212 for Cu-labeled ESBA212: ESBA212 was coupled to a CPTA chelator which binds to the isotope (macrocycle about 200Da containing four N atoms) (the 64Cu Half-life is 12.7 hours). This chelator was bound to a lysine residue in ESBA212. Since this bond is relaxed, not all lysines are labeled. Mass measurement showed that an average of 2 CPTA molecules bound to each ESBA212 molecule.

Example 13: Ex vivo labeling of plaques with ⁶⁴ Cu-ESBA212 Immunohistochemical staining was performed on fixed or non-fixed SwePS1 mouse brain sections to determine if 64Cu-ESBA212 could still bind to amyloid plaques. ^{Since 64} Cu-ESBA212 could still bind to amyloid plaques on fixed and non-fixed brain sections, ⁶⁴ Cu labeling did not change the binding properties of ESBA212 (FIG. 18).

Example 14: In vivo labeling of plaques with ⁶⁴ Cu-ESBA212 Transgenic SwePS1 mice were used to examine the binding properties of non-radiolabeled Cu-ESBA212 in vivo. Mice were applied intranasally with either 210 ug ESBA212 or 100 ug Cu-ESBA212. The animals were sacrificed 24 or 48 hours after application and the brain was analyzed for binding of ESBA212 and Cu-ESBA212 to amyloid plaques, respectively.

  As can be seen in FIG. 19, ESBA212 could be detected 24 and 48 hours after application. The same result was seen for Cu labeled ESBA212. Thus, the binding properties of Cu-ESBA212 did not change in vivo compared to ESBA212, as already demonstrated for ex vivo labeling.

Example 15: Elimination by the kidney To obtain some information about the elimination pathway of ESBA212, scFv was injected intravenously into mice. The animals were sacrificed shortly afterwards and the kidneys were analyzed by immunohistochemical methods.

ESBA212 is most likely to be filtered into the early urine in the kidney and reabsorbed in the proximal tubule, where it is degraded by the lysosomal pathway (FIG. 20).
Example 16: Marking of plaques in vivo Yet another test similar to the test performed in Example 11 was performed by intranasal administration.

Transgenic SwePS1 mice were treated intranasally with 200 μg ESBA212. Animals were perfused for 1 hour after intranasal treatment. Aβ staining of fixed SwePS1 mouse brain sections was performed and recorded as follows:
Stained with anti-His antibody (FIG. 21A);
Control stained with 6E10 (Figure 21B);
Negative control stained with anti-rabbit Cy3 (FIG. 21C);
Control stained with 22c4 IgG (FIG. 21D).

Example 17:
APPswe / PS1ΔE9 mice were treated with PBS and 22c4 IgG once a week for 3 months and twice weekly with ESBA212 for 3 months. When the treatment was completed, the mice were perfused, the brain was removed and divided into two parts. One part was homogenized, several extracts were prepared, and the levels of brain Aβ40 and 42 were measured by a commercially available ELISA test (The Genetics Company). It was observed that the levels of Aβ40 and 42 increased in the soluble fraction (TBS) and decreased in the insoluble fraction (GuHCl). In the membrane bound fraction (TBS-Triton), Aβ40 levels did not change, whereas Aβ42 levels increased (see FIG. 22). This points to Aβ redistribution from the insoluble fraction to the soluble fraction.

  The other part was used for histological examination. Paraffin sections of treated APPswe / PS1ΔE9 mice were stained with 6E10. The number and area of the plaques were evaluated by ImageJ software.

  This experiment revealed that animals treated with ESBA212 showed a significant reduction in the number and size reduction of amyloid plaques (see FIG. 23). This further supports the idea that after ESBA212 binds to Aβ fibrils, they break down into monomers or low oligomers, which shifts the equilibrium towards the soluble Aβ pool.

  The results presented above support the finding that ESBA212 enters the brain of transgenic mice after intravenous and intranasal application and binds to cortical and hippocampal Abeta plaques. Furthermore, APPswe / PS1ΔE9 mice treated intranasally with scFv showed a decrease in the number and average size of cortical amyloid plaques and a decrease in Aβ42 levels in the insoluble fraction of brain extracts.

  While presently preferred embodiments of the invention have been presented and described, it should be clearly understood that the invention is not limited thereto and that various other embodiments and implementations are possible within the scope of the claims. It is.

  References

Claims (18)

A humanized, isolated single chain variable fragment (scFv) antibody that selectively binds in vivo to the C-terminal portion of beta-amyloid, comprising:
(I) SEQ ID. No. 7 variable light chain fragment (V _L ) sequence, and (ii) SEQ ID. No. An antibody comprising a variable heavy chain fragment (V _H ) sequence of 17 sequences. 2. The scFv antibody of claim 1, which prevents beta-amyloid oligomerization; and / or mediates fibrillar beta-amyloid uptake by microglia. SEQ ID. No. The scFv antibody according to claim 1 or 2 , comprising 24 amino acid sequences. The scFv antibody according to any one of claims 1 to 3 , which is chemically modified. The scFv antibody according to any one of claims 1 to 4 , which is linked to a therapeutic agent. Linked to a label, scFv antibody according to any one of claims 1-5. ⁶⁴ The scFv antibody according to any one of claims 1 to 6, which is linked to Cu. A method for screening a subject for amyloidosis or Alzheimer's disease or for determining a subject's risk for the onset of amyloidosis or Alzheimer's disease, comprising using the scFv antibody according to claim 1.   The method according to claim 8, wherein an image created by PET imaging is used. Osamu neuropathy care, a medical drug composition for the prevention and / or delay of progression, compositions containing scFv antibody according to any one of claims 1 to 7.   A pharmaceutical composition for the treatment, prevention and / or delay of progression of Alzheimer's disease and / or for passive immunization against Alzheimer's disease, wherein the scFv antibody according to any one of claims 1 to 7 is used. A composition comprising. 12. A method for producing a pharmaceutical composition according to claim 10 or 11 , comprising the step of combining the scFv antibody according to any one of claims 1 to 7 with at least one suitable pharmaceutical carrier. . 12. A pharmaceutical composition according to claim 10 or 11 , suitable for intracranial or intravenous administration into the nasal, subcutaneous, subarachnoid cerebrospinal fluid. A nucleic acid encoding the scFv antibody according to any one of claims 1 to 7. Vector comprising a nucleic acid according to claim 1 4. Host cell comprising the vector according to the nucleic acid or the claims 1 5 according to claim 1 4. A method for producing an scFv antibody according to any one of claims 1 to 7, comprising:
(I) the host cell of claim 16 is cultured under conditions capable of synthesizing an scFv antibody;
(Ii) A method comprising recovering an scFv antibody from the culture. A test kit comprising the scFv antibody according to any one of claims 1 to 7.

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